Host factors play essential roles in modulating viral replication and pathogenesis. The important function of the host factors in flavivirus infections is highlighted by the flavivirus resistance phenotype in mice. Resistant mice can be infected by flaviviruses, but the viral liters in their tissues are 1,000-10,000 times lower than those in tissues of susceptible mice, and the spread of the infections in resistant mice is slower. The resistance to viral-induced morbidity and mortality in mice is flavivirus specific. Genetic studies demonstrated that the flavivirus resistance is controlled by a single locus as an autosomal dominant trait in mice. Recent studies suggested 2'-5'-oligoadenylate synthetase 1b (OAS1b) as a candidate gene for flavivirus-resistance phenotype. Compared with the resistance mice, susceptible mice produce an OAS1b protein lacking 30% of the C-terminal sequence, resulting in the inactivation of the OAS/RNase L pathway. Consequently, a large amount of virus is produced in the susceptible mice. Despite the recent progress, many important questions related to the molecular mechanism of the flavivirus resistance remain unknown. The objective of this application is to explore the molecular details of flavivirus resistance during West Nile virus (WNV) replication. We choose WNV as a model for our study because it is a newly emerging virus in the United States as well as a potential biodefense pathogen. Our recently developed reporting replicons and a full-length infectious clone of WNV has provided us with a unique opportunity to study WNV replication in flavivirus-resistant cells. We will accomplish the overall objective of this application by pursuing two specific aims, (i) Identify the step(s) at which WNV infection is inhibited by the flavivirus resistance gene. (II) Determine viral element(s) genetically associated with the flavivirus resistance gene. A novel approach is taken to identify potential viral element(s) that genetically associates with the flavivirus resistance gene product. Selection of adaptive WNV replicons containing an antibiotic resistance gene in the flavivirus resistant cell line is expected, for the first time, to identify viral elements genetically interacting with the flavivirus resistance gene product. The proposed work is significant, because understanding the molecular mechanism of the flavivirus resistance will provide greater insight into flavivirus replication, pathogenesis, and development of flavivirus vaccine and antiviral therapy.